ES2264438T3 - HAEMOPHILUS INFLUENZAE EXTERNAL MEMBRANE PROTEIN AND ITS USE IN VACCINATION. - Google Patents

Abstract

A recombinant bacterial outer membrane protein comprising one or more surface exposed loops, which can be obtained by a method in which one or more loops exposed on the surface of a native bacterial outer membrane protein from the surface. which is derived from the recombinant bacterial outer membrane protein, they have been replaced by one or more modified loops comprising an amino acid sequence selected from the group consisting of: SEC. ID NO. 1, SEC. ID NO. 2, SEC. ID NO. 3, and SEC. ID NO. 4, or a sequence that has an identity of at least 75% to said amino acid sequence and is capable of immunologically mimicking a corresponding antigenic determining site of the main outer membrane protein (MOMP) P5 of nontypable Haemophilus influenzae, with the condition that the recombinant bacterial outer membrane protein is not identical to the amino acid sequence of the native bacterial outer membrane protein.

Description

Haemophilus influenzae outer membrane protein and its use in vaccination.

Field of the Invention

The present invention relates to newly identified Haemophilus influenzae chimeric proteins and polynucleotides encoding these proteins. The invention also relates to a method for isolating chimeric proteins and a vaccine composition for use in the treatment of Haemophilus influenzae infection.

Background of the invention

Haemophilus influenzae (Hi) is a Gram negative cocobacillus and a strict human eater. Hi strains are either encapsulated in a polysaccharide capsule, or not encapsulated and, therefore, are classified into typifiable (encapsulated) and non-typifiable (non-encapsulated) strains.

The encapsulated pathogenic strains of Hi cause mainly, but not exclusively, invasive diseases in children under six years. For example, Haemophilus influenzae type b (Hib) is a leading cause of meningitis and other invasive infections in children. There are effective vaccines against Hib infections, and they are based on the production of antibodies to the polysaccharide capsule, and, therefore, are not effective against untypifiable Haemophilus influenzae (ntHi).

Nontypable Haemophilus influenzae (ntHi) accounts for the majority of colonizing strains and, although almost never invasive, are responsible for a significant proportion of mucosal diseases, including otitis media , sinusitis, chronic conjunctivitis and exacerbation of lower or chronic respiratory tract infections. Currently, approximately 30%, and up to 62% of ntHi are resistant to penicillin. It is estimated that it is carried by approximately 44% of children and approximately 5% of adults, and may persist for months. Neither the pathogenic mechanisms nor the host's immune response have been fully defined for otitis media caused by ntHi.

Otitis media is a common disease in children under two years. It is defined by the presence of fluid in the middle ear accompanied by a sign of acute local or systemic disease. Acute signals include ear pain, ear drainage, loss of hearing, while systemic signs include fever, lethargy, irritability, anorexia, vomiting or diarrhea. The most predominant bacteria that cause the condition are Streptococcus pneumoniae and nontypable Haemophilus influenzae (ntHi), representing 25% -50%, and 15% -30% of the cultivated species, respectively. Another common cause of the disease is Moraxella catarrhalis . In addition, ntHi is responsible for 53% of recurrent otitis media . Approximately 60% and 80% of children have at least one episode of the disease at one and three years of age respectively (the maximum being around 10 months).

It is shown that there is protective immunity for ntHi, however, antigenic deviation in epitopes naturally involved (outer membrane proteins P2, P4, P6) plays a major role in the capacity of ntHi to bypass host immune defenses.

Therefore, there is a need for additional effective vaccines against Haemophilus influenzae , and especially for vaccines against nontypable Haemophilus influenzae that is not affected by currently available Hi polysaccharide vaccines.

The Main Protein of the External Membrane (MOMP) P5 is a heat-modifiable outer membrane protein of H. Influenzae . P5 can play a role in the pathogenesis of ntHi in the form of adhesin by binding to respiratory mucin or respiratory epithelial cells infected with RSV (Reddy et al. (1996) Infect. Immun. 64: 1477-1479; Jiang et al. (1999 ) Infect Immun. 65: 1351-1356). This binding activity could be mediated through the regions exposed on the surface of the protein. Protein has been shown to be a protective antigen in several models.

There are conflicting reviews regarding the structure of this protein. Although it has been reported that the protein adopts a fibric structure consisting of assembled rolled helices, this contradicts the similarity of sequences observed between P5 and OmpA of E. coli , which is a protein that forms a β? Barrel of eight strands with four loops exposed on the surface (Munson et al. (1993) Infect Immun. 61: 4017-4020).

During persistent ntHi infections in patients with chronic bronchitis, variant strains of ntHi appear with alterations in their P5 OMP sequences. Likewise, isolates from different anatomical sites show such variability. However, this variability is limited to 4 regions for the most part. These regions correspond to the regions planned as those exposed on the surface and as a consequence that could be exposed to the pressure of the immune system. Following infection, the appearance of variants of P5 strains could be an escape mechanism for ntHi or could allow bacteria to colonize different anatomical sites (Webb and Cripps (1998) J. Med. Microbiol. 47: 1059-1067; Duim and col. (1997) Infect. Immun. 65: 1351-1356). Even so, it has been shown that the purified anti-P5 antibodies of the mice were bactericidal for homologs and a few strains of heterologous ntHi (Quigley-Reape et al. (1995) Abstr. E70, p. 239. In Abstracts of the 95th ASM general meeting
nineteen ninety five).

LB1 (f) is a 19 amino acid peptide derived from the MOMP P5 sequence from the strain of ntHi1128 (occupying the region Arg117 to Gly135). This peptide was defined as the third exposed loop of P5, and as a B cell epitope potential, by analysis of the primary sequence of P5. The immunization of animals with fimbrine chimeric peptides (called LB1 peptides), comprising: the LB1 peptide (f); a peptide bond; and an epitope of T cells, induces a protective immune response to MOMP P5 and reduces colonization of ntHi in animals subsequently exposed to ntHi (see US 5843464).

The problem of using protein antigens from only one strain of H. Influenzae in a vaccine is that the protection conferred tends to be limited largely to a homologous exposure [Bakaletz et al. (1997) Vaccine 15: 955-961; Haase et al. (1991) Infect. Immun 59: 1278-1284; Sirakova et al. (1994) Infect. Immun 62: 2002-2020]. The antigenic diversity of ntHi External Membrane Proteins means that the development of an effective vaccine in general against a group of organisms as heterogeneous as ntHi will need a new strategy.

WO 99/64067 describes a more effective use of the LB1 (f) peptide in the form of a vaccine against a broad spectrum of heterologous strains of Haemophilus influenzae expressing MOMP P5 (or naturally occurring protein variants). This involved the identification of the 3 antigenic groups of LB1 (f) peptides that define the population of the LB1 (f) peptides present in hetologous proteins of the MOMP P5 of ntHi. Chimeras of these peptides were proposed in the form of an immunogen, in order to obtain a protective immune response against a wide variety of ntHi strains.

A problem that exists is that for these peptides work optimally as effective immunogens, they must be trained to generate antibodies that recognize and unite the epitopes in their native structure.

As a consequence of this, the present invention refers to a procedure to increase the effectiveness of peptides LB1 (f) introducing them in loops exposed in the surface of other outer membrane proteins, or, preferably, back to MOMP P5 itself, such that the immune system can better recognize epitopes in their native conformation. Such outer membrane proteins Recombinants of the invention possess one or more of the following advantages: the context of the important LB1 (f) B epitopes is found in a favorable context (with a restricted structure) for better immune recognition and immunogenicity; the epitopes LB1 (f) protectors can replace hypervariable epitopes, non-protective outer membrane protein to center the immune response to protective LB1 (f) peptides; the recombinant outer membrane protein, modified, helps provide a better protective immune response against a wide range of ntHi strains.

Summary of the invention

An object of the present invention is provide a recombinant outer membrane protein consisting of one or more loops exposed on the surface, in the that one or more native loops exposed in the protein surface (those loops present in the protein native, wild type) by one or more modified loops that comprise an amino acid sequence selected from a group constituted by: SEQ. ID NO: 1-8 (each constituted by a peptide LB1 (f)), or variants of said antigenically related sequences, which have a identity of at least 75% and are able to imitate immunologically a corresponding antigenic determining site of the MOMP P5 of ntHi.

Preferably, the recombinant outer membrane protein is derived from a native (wild-type) outer membrane protein that comes from non-typable Haemophilus influenzae or Moraxella catarrhalis . This is advantageous in providing other protective antigens against H. Influenzae in an individual molecule, or to provide an individual molecule that can provide protection to a host against two causes of otitis media: ntHi and Moraxella catarrhalis .

In a preferred embodiment, a MOMP P5 protein modified, recombinant. The advantage of such proteins is that they already contain an LB1 (f) peptide in the third loop, in a protective, native conformation (which preferably should not be changed). Of course, in the present embodiment, in which only modifies a native (individual) loop by replacing it with a loop consisting of a peptide LB1 (f), the invention does not cover embodiments in which the individual native loop is the third loop of the native OMP (which already comprises a peptide LB1 (f)).

Another object is to provide polynucleotides that encode such proteins. The invention also relates to a method for isolating proteins, and a vaccine composition for use in the treatment of Haemophilus influenzae infection, or otitis media.

The invention can be understood more Complete by reference to the following drawings and description detailed.

Brief description of the drawings

Figure 1: The MOMP amino acid sequence P5 A conservative evaluation of the position of the 4 is indicated external loops Loop 3 corresponds to an LB1 (f) peptide of Group 2b.

Figure 2: Part topology model integrated of the MOMP P5 outer membrane from a strain of nthi 1128 (loop 3 corresponds to a peptide LB1 (f) of Group 1). When looking from left to right along the outer surface of the outer membrane, it is likely that the border residues A, F, D, G, A, G, W and C relate to the outer membrane, and therefore, the 4 amino acid sequences outside of, but not including, the aforementioned border residues previously, they are a liberal (and more accurate) assessment of the position of the 4 external loops.

Detailed description of the preferred embodiment Proteins of the External Membrane of the Invention

The recombinant outer membrane protein of the invention can be obtained from any bacterial, native (wild-type) outer membrane protein, which has at least one surface exposed loop. Preferably, said native outer membrane protein should be derived from a Gram negative bacterium, more preferably from Haemophilus influenzae (preferably non-typable H. influenzae ) or Moraxella catarrhalis .

In Genbank, or in WO 96/33276, DNA sequences of various proteins of the outer membrane of H. influenzae are known and described, for the purpose of the present invention. The information in such descriptions will allow those skilled in the art to clone the native gene from ntHi. For the purposes of the present invention, preferred ntHi native outer membrane proteins are: P1 (Bolduc et al. 2000 Infect. Immun. 68: 4505); P2 (Sikkema and Murphy (1992) Infect. Immun. 60: 5204; Kyungcheol and Murphy (1997) Infect. Immun. 65: 150; Neary et al. (1999) 99th Gen. meeting of the ASM, Poster E-10. ; Duim et al. (1996) Infect. Immun. 64: 4673): P4; P5 (WO 94/26304); D15 (WO 94/12641); Omp26 (WO 97/01638); HMW [1 & 2] (Barenkamp and Leininger (1992) Infect. Immun. 60: 1302; Loosmore et al. WO 00/20609; Loosmore et al. WO 00/35477; Barenkamp document WO9736914A); HMW [3 & 4] (Barenkamp et al. WO9736914-A1); HxuA (Cope et al. (1994) Mol. Microbiol. 13: 863 .; Hanson et al. (1992) PNAS 89: 1973.); ..HgpA (Jin et al. (1996) Infect. Immnu. 64: 3134; Hanson et al. (1992) Infect. Immun. 60: 2257; Jin et al. (1999) Microbilogy 145: 905.); TbpA (Loosmore et al. US 6008326; Loosmore et al. US 6015688); Hsf / Hia (Loosmore et al. WO 00/55191; Barenkamp and StGemelII (1996) Mol. Microbiol. 19: 1215.); Hap (StGeme et al. (1994) Mol. Microbiol. 14: 217.); lomp1681 (GB 0025998.6); D15b (WO 00/47737); HasR (WO 00/50599); YadA; IgAprotease; and VirG (GB 0026002.6).

In Genbank, or in WO 00/78968, the DNA sequences of several proteins of the outer membrane of Moraxella catarrhalis are known and described, for the purpose of the present invention. The information in such descriptions will allow those skilled in the art to clone the native gene from Moraxella catarrhalis . For the purposes of the present invention, the preferred native proteins of the outer membrane of Moraxella catarrhalis are: OmpA; OmpB1 / B2 (Chen et al. WO 98/33814); OmpCD (Hsiao et al. (1995) Microb. Pathog. 19: 215.) OmpE (Murphy et al. US 5948412); Omp106 (WO 97/41731 and WO 96/34960); TbpA (WO 97/13785 and WO 97/32980); LbpA (WO 98/55606); CopB (Helminen et al. (1993) Infect. Immun. 61: 2003-2010); OmplA 1 (WO 00/15802); D15 (WO 99/63093); D15b (document 00/52042); PilQ (WO 99/64448); Mip (WO 00/09694); HasR (WO 99/64602); OmpA (WO 00/71724); AperE (GB 9912038.8); OmpF (GB 9912674.0); OmpS (GB 9912705.2); HutA1 / A2 (GB 9912838.1 / GB 9913354.8); FHA C (GB 9921693.9); PorA (document PCT / EP00 / 09034); CyaE (GB 9922829.8); UspA1 and UspA2 (WO 93/03761); and Omp21.

Recombinant outer membrane proteins of the invention

Those skilled in the art are perfectly trained to determine loops exposed on the surface of native outer membrane proteins using models of methodology and topology that are already known for the proteins of the outer membrane indicated above. Typically, experts in the technique would determine where these loops are running secondary structure prediction programs looking for β strands (secondary structure that crosses the membrane external for bacterial OMP). The β strands of the OMP that cross the membrane tend to have a length of approximately 10 amino acids, and tend to be amphipathic. They are usually searched pairs of β strands (in total at least 2 β strands, but sometimes up to 20 or so), as the OMP tend to start and finish on the inside of the outer membrane. With frequency, aromatic amino acids trace the principle and / or the End of such a thread. Loops exposed on the surface reside between pairs of β strands. Other indications that it has been identified an exposed loop on the surface are that: a) tend to be longer than the loops of the inner surface of the membrane (5 to 30 or more amino acids versus 2-6 amino acids), and b) tend to be quite variable when compare the same sequence in different strains of the same bacteria (while the sequences of the β strands tend to be preserved) Preferably, loops exposed on the surface are determined using topology models tested in a way Experimental (or structures) of outer membrane proteins homologous (with amino acid identity greater than 20, 40, 50, 60, 70, 80 or 90%). There are also experimental tests for determine the loops exposed on the surface: the loops are the protein fragments that activate antibodies in a host (and the antibodies so collected can be used to determine where surface epitopes are found in the primary sequence of the protein); and protein areas likely to be processed by proteases.

For example, the 4 loops exposed (so external) on the surface of MOMP P5 (of ntHi) are indicated in the Figures 1 (a conservative topology for an LB1 (f) of Group 2b of MOMP P5) and 2 (a more accurate representation of the topology of MOMP P5 loops for a LB1 (f) of Group 1 of MOMP P5), and those skilled in the art can determine them Perfectly for other variants of MOMP P5. This is because regions related to the outer membrane (as shown in Fig. 2) they are very well conserved among all the proteins of MOMP P5. Therefore, looking from left to right along the outer surface of the outer membrane of Figure 2, is it is probable that the border residues A, F, D, G, A, G, W and C are relate to the outer membrane, and therefore the 4 sequences of amino acids outside, but not including, residues boundary mentioned above, are an accurate assessment of the position of the 4 external loops.

LB1 (f) peptides are constituted from 13 to about 22 amino acids. Peptides form 3 main immune groups: 1, 2 and 3 (dividing the group 2 in two slightly different subgroups: 2a and 2b). At WO 99/640647 describes a broad set of peptides LB1 (f) known from the 3 groups (and their variants).

A surface exposed loop is replaced (native) by a loop exposed on the modified surface if the wild-type surface loop is altered from any form, so that it contains an LB1 (f) peptide. Therefore the term covers where an LB1 (f) peptide is inserted (or placed inside) the target loop at any position of the native loop. Preferably, the insertion is at the center point of the loop. The replacing a loop can be a complete sequence change of the native loop, or a partial change. A partial change can be of 1, 2, 3, 4, 5 or more amino acids of a loop, and is preferably a continuous sequence of amino acids in the loop. Preferably the complete loop is replaced, or the complete loop except 1-2 amino acids at each end of the loop. A loop of X amino acids can be substituted for an X amino acid peptide for optimal folding of the outer membrane protein recombinant

The modified loops of the invention should comprise an LB1 (f) peptide. These loops are modified in terms of being a non-native environment in the recombinant outer membrane protein of the invention (therefore, the modified loop may be a wild-type sequence of loop 3 of MOMP P5). As described in WO 99/64067, the LB1 (f) peptide groups contain a wide variety of sequences of immunologically related variants, which have an identity of at least 75% with the representative peptide of the group shown in SEQ ID NO: 1-4. More preferably, the loop exposed on the native surface should be completely replaced by a complete (preferably native) loop 3 of MOMP P5. It is more likely that such substituted loops adopt the native conformation of the LB1 (f) epitope of loop 3 within the altered outer membrane protein. SEQ ID NO: 5-8 shows examples of full loop 3. Those skilled in the art can determine them perfectly by comparing a sequence of MOMP P5 with the topology model of Fig. 2. The modified loops are preferably 13-75 amino acids in length, more preferably 15-50, still more preferably 20 -40, and more preferably a length of about 30 amino acids
two.

LB1 (f) peptides refer to representative peptides of Groups 1, 2a, 2b and 3 (SEQ ID NO: 1, 2, 4 and 3 respectively, comprised within the complete loop 3 sequences of SEQ ID NO: 5, 6, 8 and 7 respectively), and antigenically related variants of these peptides (or complete loop 3 sequences). The "antigenically related variants" may be natural variants (as illustrated by the peptides described in WO 99/64067) or artificially modified variants that immunologically mimic the LB1 (f) antigenic determinant site of the MOMP P5 protein. The antigenically related variants of the peptides should have an amino acid sequence identity of at least 75% to one of the peptides provided in SEQ ID NO: 1-8 (and more preferably at least 85%, and more preferably a identity of at least 95%), while still being able to immunologically mimic the corresponding antigenic determinant site of MOMP P5 of untypifiable Haemophilus influenzae . For the present invention, "immunologically mimicking the corresponding antigenic determinant site of the MOMP P5 of ntHi" is defined as a peptide (variant) (or complete loop 3) inserted or substituted in a loop of an outer membrane protein that is capable of inducing antibodies that specifically recognizes one of the wild-type LB1 (f) sequences (listed in Tables 2, 3 and 4 of WO 99/64067) in the context of their natural environment within the MOMP P5 Y / O it is defined as a peptide (variant) (or complete loop 3) inserted or substituted in a loop of an outer membrane protein that is capable of being recognized by the same immunospecific antibody that recognizes one of the sequences of LB1 (f) of wild type (listed in tables 2, 3 and 4 of WO 99/64067) in their natural context within the MOMP-P5 protein. Preferably, the recognition test used above is that a sequence (wild type or variant) has more than 30, 40, 50, 60, 70, 80, 90% of the avidity of the other sequence (wild type or variant, respectively) in an ELISA assay using the antibodies as described above. More preferably, the variant sequence is approximately equivalent to the wild-type sequence in terms of being able to protect a host against untypifiable H. influenzae .

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It may have been added, inserted, replaced or Amino acids removed in antigenically related variants.  Preferred variants are those that differ from those references for conservative amino acid substitutions (preferably individual).

Those skilled in the art can achieve such peptide insertions and substitutions using conventional and well-known molecular biology techniques (see, for example, the conventional Sambrook et al. Molecular Cloning a Laboratory Manual (1989) Cold Spring Harbor Laboratory Press) . In particular, knowing the DNA sequence of the target of the native outer membrane protein, primers may simply be designed to replace a nucleotide sequence encoding a native loop sequence with a nucleotide sequence encoding a loop sequence. modified by PCR.

In a preferred embodiment, the native outer membrane protein target is MOMP P5 itself. In Fig. 2 the sequences of loops 1, 2, 3 and 4 can be observed. Surprisingly, the modified MOMP P5 proteins of the invention have one or more of the following advantages: the LB1 (f) peptides are located in a most favorable position to assume a natural conformation for an effective optimal immune response against the peptide in its natural environment; non-protective, highly variable loop structures (in loops 1, 2 and 4) can be eliminated to center the immune response outside these epitopes; an individual immunogenic MOMP P5 molecule can be made that can provide a host with protective immunity against a wide range of ntHi strains; The grouping of LB1 (f) peptides into the different loops of an individual molecule provides a synergistic improvement of a host's immune response against a wide range of strains of
ntHi.

Preferably, the third protein loop remains intact This is advantageous because this loop is already carries a peptide of LB1 (f) in a native conformation.

Preferably, in the MOMP P5 protein modified one or more may have been substituted (preferably all) loops 1, 2 and 4 for a modified loop comprising a LB1 (f) peptide different from Group 1, 2a, 2b or 3 (in any order), which is also from a group other than the peptide LB1 (f) retained in loop 3. For example, if the peptide of loop 3 is from group 3, native loops 1, 2 and 4 can replaced by a modified loop comprising a peptide of the Group 1, 2a and 2b, respectively (or in fact in any order), or its antigenically related variants. Preferably, it select the LB1 (f) peptides from the group SEQ ID NO: 1-4 (which contains a representative from each group of peptides) If a complete native loop is replaced by a complete loop sequence 3, preferably the loop sequences 3 of the group SEQ ID NO: 5-8 (which It contains a representative from each group of LB1 (f)).

Alternatively, in the modified MOMP P5 protein one or more (preferably both) of loops 1 and 2 respectively have been replaced by a modified loop comprising an LB1 (f) peptide other than Group 1, 2a, 2b or 3 (in any order), which is also from a Group other than the LB1 (f) peptide retained in loop 3, and loop 4 is replaced by a modified loop comprising another protective epitope of H. Influenzae . Said other epitope is preferably the 5 or 6 loop peptide of the MOMP P2 of ntHi. Preferably, the complete loop 4 is replaced by a modified loop which is the sequence of the MOMP P2 peptide of the complete loop 5 or 6 (topology models for MOMP P2 are known in the art that clearly define the regions of the loop: Kyungcheol and Murphy (1997) Infect. Immun. 65: 150, Neary et al. (1999) 99th Gen. meeting of the ASM, Poster E-10 .; Duim et al. (1996) Infect. Immun. 64: 4673). Preferably, the LB1 (f) peptides are selected from the group SEQ ID NO: 1-4. If a complete native loop is replaced by a complete loop sequence 3, preferably the loop sequences 3 of the group SEQ ID NO: 5-8 (containing a representative of each group of LB1 (f)) are selected.

In addition, the truncation of the proteins of the MOMP P5 of the invention, which have all 4 regions of loops still intact, they are also considered proteins of the invention.

Polynucleotides of the invention

Another aspect of the invention is a molecule of DNA or RNA that encodes an outer membrane protein recombinant of the invention. To establish this, degeneration Codon usage is relevant. Preferably, so that well-known codons are optimal for expression, they should use guests in different expressions. Preferably, the nucleotides encoding LB1 (f) peptides have the same sequence that the wild type sequences arranged in the Tables 6-8 of WO 99/64067.

The invention also provides polynucleotides. which are complementary to the polynucleotides described above.

When the polynucleotides of the invention are used for recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading phase with other coding sequences, such as those encoding a guide or secretory sequence, a pre-, or pro-preproprotein sequence, or other fragments of fusion peptides. For example, a tag sequence that facilitates purification of the condensed polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the tagged sequence is a hexa histidine peptide, as provided in the pQE vector (Qiagen, Inc) and described in Gentz et al., Proc. Natl Acad. Sci . USA 86: 821-824 or is a brand of HA, or is glutathione - S-transferase. The polynucleotide may also contain 5 'and 3' non-coding sequences, such as transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

Vectors, host cells, expression

Still other aspects of the invention are a expression vector comprising the DNA or RNA molecule of the invention, wherein said expression vector is capable of expressing a recombinant outer membrane protein of the invention when present in a compatible host cell, and a cell host comprising this expression vector.

An alternative embodiment is a guest recombinant containing the DNA or RNA molecule of the invention inside your chromosome (which can be perfectly integrated through of well known techniques, such as homologous recombination using a nucleotide sequence known in the genome), in the that said molecule is in a context suitable for express a recombinant outer membrane protein from the invention.

For recombinant production, host cells can be engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. The introduction of polynucleotides into host cells can be accomplished by procedures described in many conventional laboratory manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY , (1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL , 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), such as calcium phosphate transfection, DEAE-dextran-mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, charge by curettage, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such as meningococcal, streptococcus, staphylococcus, E. coli, streptomyx and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9 cells ; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. Preferably, the host is ntHi or Moraxella catarrhalis when the DNA molecule of the invention is integrated into the genome of the host cell.

A wide variety of expression systems can be used. Such systems include, among others, systems derived from chromosomes, episomes and viruses, for example, vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papova virus, such as SV40, vaccinia virus, adenovirus, avian smallpox virus, pseudorrabia virus and retrovirus, and vectors derived from combinations thereof, such as the derived plasmid and bacteriophage genetic elements, such as Cosmids and phagemids. Expression systems may contain control regions that regulate as well as generate expression. Generally, any suitable system or vector can be used to maintain, propagate or express polynucleotides to produce a polypeptide in a host. The appropriate nucleotide sequence can be inserted into an expression system by any of a variety of well known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A MANUAL LABORATORY , (above).

Purification of expressed polypeptides / peptides recombinant

Yet another aspect of the invention is a process for producing a recombinant outer membrane protein of the invention, which comprises culturing the host cell (containing the DNA molecule of the invention within an expression vector, or integrated into its chromosome) under conditions sufficient for the production of said protein, and the recovery of the recombinant outer membrane protein. The protein can be recovered in the form of a purified product. The protein can also be recovered within a blister preparation (outer membrane vesicle) that can be generated from the host cell (particularly when there is a Gram negative bacterium, preferably ntHi or M. catarrhalis ) using known techniques. The protein can be recovered within a phantom preparation (outer membrane) that can be generated from the host cell (in particular, when there is a Gram negative bacterium, preferably ntHi) using known techniques (see WO 92/01791). Finally, the protein can be recovered within a preparation of whole dead, living or attenuated whole cells from the host bacteria.

It exists within the general common knowledge of those skilled in the art how to isolate vesicles or ghosts from the outer membrane from a host that would contain the protein of the invention. The advantage of such techniques is that the protein of the recombinant outer membrane of the invention remains properly folded inside the vesicles and thus presents its native conformation to the immune system if used as an immunogen.

The proteins of the invention can be recovered. and purified from recombinant cell cultures to through well-known procedures, including precipitation by ammonium sulfate or ethanol, acid extraction, anionic or cation exchange chromatography, chromatography on phosphocellulose, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lecithin chromatography Well-known techniques to replicate proteins can be used to regenerate active conformation when the polypeptide is denatured during isolation and / or purification.

Although the gene sequence of the chimeric LB1 (f) polypeptide in the vector can be labeled with a Histidine branded sequence that helps purification of the polypeptide, it is not an essential element for the invention, since that polypeptides can still be purified without the mark of Histidine through one of the mentioned techniques previously.

Vaccinations

Another aspect of the invention is a composition. of vaccine (or an immunogenic composition) comprising a immunogenic amount (preferably an effective amount or protective) of the recombinant outer membrane protein of the invention (or isolated or purified, or present in a preparation of vesicle of the outer membrane, ghost or whole cells dead, alive or with attenuated life) in an excipient pharmaceutically acceptable, and an optional adjuvant. In this context, the immunogenic amount is defined as an amount enough of protein to elicit an antibody response in a host vaccine

Other aspects of the invention are still: the use of an immunogenic amount of recombinant outer membrane protein of the invention in a pharmaceutically acceptable excipient, and an optional adjuvant, to prevent or treat Haemophilus influenzae disease (preferably otitis media , sinusitis , conjunctivitis or lower respiratory tract infection); a method for inducing an immune response in a mammal susceptible to Haemophilus influenzae infection which comprises administering an effective amount of the above-mentioned vaccine to the mammal (an amount being effective capable of protecting a host [such as chinchilla ] to some extent against a ntHi infection); and a method for preventing Haemophilus influenzae infection comprising administering an effective amount of the vaccine of the invention to a mammal.

The vaccines of the invention are capable of provoke a cross-protection immune response against a large variety of ntHi strains (particularly when integrated one or more modified loops in an outer membrane protein from ntHi).

A preferred vaccine of the invention comprises a modified M. catarrhalis outer membrane protein, comprising one or more modified regions of loops, since such preparations can protect a host more effectively against otitis media by immunization with an individual molecule. .

In general, vaccine preparation is described in Vaccine Design (The subunit and adjuvant approach (publishers Powell MF & Newman MJ). (1995) Plenum Press New York).

Additionally, the proteins of the present invention are preferably adjuvant in the formulation of vaccines of the invention. Suitable adjuvants include salt of aluminum such as aluminum hydroxide gel (alum) or phosphate of aluminum, but they can also be a salt of calcium, iron or zinc, or they can be an insoluble suspension of acylated tyrosine, or acylated sugars, cationic derivatized polysaccharides or anionically, or polyphosphazenes. Other known adjuvants include oligonucleotides containing CpG. Oligonucleotides are characterized in that the CpG dinucleotide is unmethylated. Such oligonucleotides are well known and are described in, for example, WO 96/02555.

Other preferred adjuvants are those that induce an immune response, preferably of the TH1 type. The high levels of cytokines of type Th1 tend to favor induction of cell-mediated immune responses to the antigen provided while elevated cytokine levels of Th2 type tend to favor the induction of immune responses humoral to the antigen. Appropriate adjuvant systems include, for example, monophosphoryl lipid A, preferably monophosphoryl lipid A 3 de-O-acylated (3D-MPL), or a combination of 3D-MPL together with an aluminum salt. The CpG oligonucleotides also preferably induce a TH1 response. An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative, especially the combination of QS21 and 3D-MPL, as described in WO 94/00153, or a less reagenic composition, in which QS21 is inactive with cholesterol, as described in WO 96/33739. In WO 95/17210 a formulation of particularly potent adjuvant that involves QS21 3D-MPL and tocopherol in an oil emulsion in water, and is a preferred formulation.

The vaccine composition of the invention is preferably administered orally, intranasally or parenterally (including subcutaneous, intramuscular, intravenous injection, intradermal, transdermal). The right formulations for parenteral administration include injection solutions sterile aqueous and nonaqueous which may contain antioxidants, buffers, bacteriostatic and solute compounds that make the isotonic formulation with the recipient's blood; and suspensions sterile aqueous and non-aqueous which may include agents of suspension or thickening agents. The formulations can be present in unit dosage or dosage containers multiple, for example, sealed ampoules and vials and can be store in a freeze-dried condition that only requires the addition of the sterile liquid vehicle immediately before use. The vaccine formulation may also include adjuvants as described above. The dose will depend on the specific activity of the vaccine and can be determined perfectly through routine experimentation. It should be a amount that induces an immunoprotective response without effects Adverse and significant side effects in typical vaccines (typically 1-100 µg of protein antigens, preferably 5-50 µg, and more typically in the interval 5-25 \ mug).

However, another aspect relates to a vaccine / immunological formulation comprising the polynucleotide of the invention. Such techniques are known in the art, see, for example, Wolff et al. Science , (1990) 247: 1465-8.

The proteins of the present invention can be administered in the form of multivalent subunit vaccines in combination with antigens of other H. influenzae proteins to achieve enhanced bacterial activity. They can also be administered in combination with polysaccharide antigens, for example, the PRP polysaccharide capsule (preferably conjugated to a protein such as tetanus toxoid) of H. influenzae b. For administration in combination with epitopes of other proteins, the protein of the invention can be administered separately, in the form of a mixture (for example within a vesicle preparation of the outer membrane) or in the form of a conjugate or genetic fusion polypeptide. The conjugate is formed through conventional techniques to couple protein materials. The proteins of the invention can be used together with antigens of other organisms (for example, encapsulated or non-encapsulated, bacteria, viruses, fungi and parasites). For example, the proteins of the invention are useful together with antigens of other microorganisms involved in otitis media and other diseases. These include Streptococcus pneumoniae , Streptococcus pyrogenes group A, Staphylococcus aureus , respiratory syncytial virus and Moraxella catarrhalis .

The evaluation of the proteins of the invention as potential vaccines against otitis media caused by ntHi can be performed in an animal model of chinchilla (WO 99/64067). This model mimics the development of otitis media in children and is based on the successive intranasal administrations of adenovirus and ntHi in a separate week. In these conditions, after colonization of the nasopharynx, the bacteria is able to invade the middle ear through the eustachian tube. Once there, ntHi will proliferate and induce an inflammatory process similar to that seen in children.

For the evaluation of vaccines, by then chinchilla has been actively immunized, they are too old at the time of exposure to be inoculated intranasally with ntHi: even with a pre-infection with adnovirus, none of them will develop otitis media . As an alternative route of exposure, a direct inoculation of the bacteria in the middle ear (bulla) through the skull is used. Passive transfer / exposure protocols can also be used to avoid necessary transbulbar exposures.

With all these types of exhibitions, you can highlight the severity of the disease by observation otoscopic (through the outer ear) or tympanometry, which evaluates the level of inflammation in the middle ear or changes in pressure of the middle ear and presence of fluid in the middle ear, respectively. The efficacy of a vaccine is determined by the reduction of severity and / or duration of inflammation and reduction of colonization in the ear and nasopharynx.

The vaccines of the invention can be further evaluated by examining whether the proteins of the invention inhibit the adhesion of ntHi to the epithelial cells of the throat of the chinchilla, and whether they can prevent colonization of the nasopharynx by ntHi in vivo . Colonization of the nasopharynx is an initial stage required for the development of otitis media , therefore, this inhibition of colonization will also help to inhibit the development of otitis media .

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{} \ hskip1cm Jan Poolman

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one

Claims (16)

1. An outer membrane protein recombinant bacterial comprising one or more loops exposed in the surface, which can be obtained by a procedure in the that one or more loops exposed on the surface of a protein the native bacterial outer membrane from which it is derived the recombinant bacterial outer membrane protein, have replaced by one or more modified loops comprising a amino acid sequence selected from the group constituted by: SEC. ID NO. one, SEC. ID NO. 2, SEC. ID NO. 3, and SEC. ID NO. 4, or a sequence that has an identity of at least 75% to said amino acid sequence and is capable of immunologically mimicking a corresponding antigenic determining site of the main external membrane protein (MOMP) P5 of Haemophilus influenzae not typifiable, with the condition that the recombinant bacterial outer membrane protein is not identical to the amino acid sequence of the native bacterial outer membrane protein. 2. The outer membrane protein recombinant bacterial of claim 1, which can be obtain by a procedure in which one or more loops exposed on the surface of an outer membrane protein native bacterial from which the protein is derived from the recombinant bacterial outer membrane, has been replaced by one or more modified loops comprising an amino acid sequence  selected from the group consisting of: SEC. ID NO. 5, SEC. ID NO. 6, SEC. ID NO. 7, and SEC. ID NO. 8, or a sequence that has an identity of at least 75% to said amino acid sequence and is capable of immunologically mimicking a corresponding antigenic determining site of the M5P P5 of non-typable Haemophilus influenzae , with the proviso that the outer membrane protein Recombinant bacterial is not identical to the amino acid sequence of the native bacterial outer membrane protein. 3. The recombinant bacterial outer membrane protein of claim 1 or 2, wherein it is obtained from a non-typable Haemophilus influenzae native membrane protein or Moraxella catarrhalis . 4. The recombinant bacterial outer membrane protein of claim 3, wherein it is obtained from the M5P P5 of nontypeable Haemophilus influenzae . 5. The modified MOMP P5 protein from claim 4, wherein loops 1, 2 and 4 have been each one substituted by a modified loop comprising a peptide LB1 (f) different selected from the group consisting of: Group 1, 2a, 2b and 3 peptides, in which the modified loops they comprise a peptide LB1 (f) that comes from a Group different from peptide LB1 (f) present in loop 3. 6. The modified MOMP P5 protein of claim 4, wherein loops 1 and 2 have each been replaced by a modified loop comprising a different LB1 (f) peptide selected from the group consisting of: Group 1 peptides, 2a, 2b and 3, in which the modified loops comprise a peptide LB1 (f) that comes from a group other than the peptide LB1 (f) present in loop 3, and loop 4 has been replaced by another protective epitope of H influenzae . 7. The modified MOMP P5 protein from claim 6, wherein the loop 4 has been replaced by a modified loop comprising a protective epitope of loop 6 of the MOPM P2. 8. The modified MOMP P5 protein from claims 4-7, wherein the loops modified comprise a modified LB1 (f) peptide selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and 8. 9. A vaccine composition that comprises an effective amount of the membrane protein recombinant bacterial external of the claims 1-8 in a pharmaceutically acceptable excipient and an optional adjuvant. 10. The use of an immunogenic amount of the recombinant bacterial outer membrane protein of claims 1-8 in a pharmaceutically acceptable excipient and an optional adjuvant in the preparation of a medicament for the prevention or treatment of Haemophilus influenzae disease. 11. The use of claim 10 wherein the Haemophilus influenzae disease is otitis media , sinusitis, conjunctivitis or lower respiratory tract infection. 12. A DNA or RNA molecule that encodes a recombinant bacterial outer membrane protein as it set forth in claims 1-8. 13. An expression vector comprising a DNA or RNA molecule of claim 12, wherein said expression vector is able to express said protein from the recombinant bacterial outer membrane when present in a compatible host cell. 14. A host cell comprising the vector of expression of claim 13. 15. A recombinant host consisting of the DNA or RNA molecule of claim 12 on its chromosome, in which said molecule is in a context suitable for express said bacterial outer membrane protein recombinant 16. A procedure to produce a recombinant bacterial outer membrane protein that comprises the culture of the host cell of claim 14 or 15 under conditions sufficient for the expression of said protein, and bacterial outer membrane protein recovery recombinant, or outer membrane vesicles or ghosts that They comprise said protein.